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Molecular genetics of bacteriophages of lactic acid bacteria
Ek Arendt, M van de Guchte, Ag Coffey, C Daly, Gf Fitzgerald
To cite this version:
Ek Arendt, M van de Guchte, Ag Coffey, C Daly, Gf Fitzgerald. Molecular genetics of bacteriophages
of lactic acid bacteria. Le Lait, INRA Editions, 1993, 73 (2), pp.191-198. �hal-00929327�
Bacteriophages
Molecular genetics of bacteriophages of lactic acid bacteria
EK Arendt, M van de Guchte, AG Coffey, C Daly, GF Fitzgerald
Department of Food Microbiology and National Food Biotechnology Centre, University College, Cork, Ireland
Summary - Bacteriophage infection of lactic acid bacteria starter cultures can result in serious dis- ruption or even failure of fermentation processes. With the help of newly developed techniques, progress has been made in the molecular characterisation of phages of lacüc acid bacteria (LAB) and also in the elucidation of the nature of their interaction with host cultures. A number of receptors involved in phage adsorption have been indentified and DNA penetration and injection, as weil as the intracellular development of the LAB phages have been investigated. The structural organisation ofLactobacillus and lactococcal phage genomes have also been determined and a number of phage genes has been cloned and sequenced. These include determinants for phage Iysin and phage structural proteins. Specific loci, attachment sites (att), involved in the Integration of temperate phage genomes, the cohesive ends(cos), involved in phage genome circularisation and the packag- ing sites (pac) of circularly permuted phages have been localised and in many cases characterised at a molecular levaI.
lactic acid bacteria 1bacteriophages 1molecular characterisation Ilysogeny
Résumé - Génétique moléculaire des bactériophages des bactéries lactiques. Les infections des cultures starter de bactéries lactiques par des bactériophages peuvent provoquer de sérieuses perturbations ou même l'arrêt du processus de fermentation. Grâce aux techniques nouvellement développées, des progrès ont été faits dans la caractérisation moléculaire des phages des bactéries lactiques, ainsi que dans la compréhension de la nature de leur interaction avec les cultures hôtes.
Plusieurs récepteurs impliqués dans l'adsorption des phages ont été identifiés et l'on aétudié la pé- nétration et l'injection de l'ADN, ainsi que le développement intracellulaire des phages des bactéries lactiques. On adéterminé l'organisation structurale des génomes de phages deLactobacilluset de Lactococcus,et de nombreux gènes phagiques ont été clonés et séquencés. Cela inclut des déter- minants de la lysine et de protéines structurales phagiques. Des loci spécifiques, sites d'attache- ment (att),impliqués dans l'intégration des génomes des phages tempérés, les extrémités cohésives (cos),impliqués dans la circularisation des génomes phagiques et les sites d'empaquetage (pac) des phages àpermutations circulaires, ont été localisés et, dans de nombreux cas, caractérisés au niveau moléculaire.
bactéries lactiques / bactériophages / caractérisation moléculaire /Iysogénie
192 EK Arendt et al
INTRODUCTION
Bacteriophage infection is known as the major cause of the inhibition of LAB starter cultures used in food fermentations. This is particularly true in the dairy industry for the following reasons: the fermentation is carried out in a non sterile-medium (pas- teurised milk); batch culture fermentations are conducted under increasingly stringent manufacturing schedules; the amount of specialized cultures available on the mar- ket is Iimited; and the continuous use of defined cultures provides an ever-present host for bacteriophage attack. Through the years, efforts have been made to identify the major species of bacteriophages of LAB, to elucidate the specific nature of their interactions with their hosts and to define their physiological and genetic traits as an aid to determining their molecular characteristics. The information obtained will provide nover approaches for the con-
Table 1. Phage receptors 01 LAB.
struction of phage resistant starter cultures and will assist in the identification of fac- tors controlling phage gene expression.
PHAGE ADSORPTION
The first step in the interaction between a lytic phage and its host occurs when the phage particle adsorbs to the cell surface.
Electron microscopie studies with lactococ- cal phages have shown that these either adsorb to their homologous hosts in small groups (5-30 phages) at evenly distributed spots or they adsorb uniformly over the cell surface (Budde-Niekiel and Teuber, 1987).
Phage receptors are generally cell wall 10- cated; an overview of phage receptors iden- tified to date is presented in table1.Recent- Iy, Valyasevi et al(1991) reported that the reversible binding to the cell wall polysac- charide is followed by an irreversible inter- action with a cell membrane located protein.
Receptor Phage Reference
Lactococcus
Lipoprotein 01 the plasma membrane L-Rhamnose,o-galactosamine o-glucosamine (cell-wall associated) Rhamnose, extracellular
polysaccharide
Carbohydrate component 01 the peptidoglycan
Lactobacillus
Rhamnose (cell-wall), o-galactosamine (cytoplasmic membrane)
L-Rhamnosyl (peptidoglycan)
0m13 Oram, 1971
1b7 Koegh and Pettingill,
1983
0kh Valysevi et al, 1990
P008,P127 Schâler et al, 1991
PI-1 Yokokura, 1977
PL-1 Ishibashi et al, 1982
DNA PENETRATION AND INJECTION
ForLactobacillus caseiphage PL-1, it has been shown that Ca2+ and ATP were es- sential for penetration of the phage DNA (Watanabe and Takesue, 1972; Watanabe et al, 1979). Watanabe et al (1991) have examined injection ofLb caseiphage PL-1 by electronmicroscopy and have demon- strated that the process was inhibited by chloramphenicol and erythromycin, both of which inhibit protein synthesis. Thus it is possible that protein synthesis is needed in the early stage of the phage infection and is necessary for complete DNA transfer and injection.
INTRACELLULAR DEVELOPMENT OF LAB PHAGES
The burst sizes and the latent periods of the LAB phages are summarised in table II. Generally, the optimum temperature for phage replication is similar to that of the host strain. Phage multiplication can also be dependent on the nutritional status of the host and on electrolyte availability, both of which can be active in promoting phage replication and cell Iysis (Keogh, 1980; Klaenhammer, 1984).
Table Il. Intracellular development of LAB phages.
Very Iittle is known about the intracellu- lar development of LAB phages at a mo- lecular Javel.Hillet al(1991a) have report- ed that the DNA molecules of the cohesive ended phage 31 are synthesized in a con- catameric form between 20-40 min after infection of L lactis NCK203. Sixty min at- ter infection, there was a decrease in the detectable amount of phage DNA, which was possibly due to packaging and release of the phage particles. Powellet al (1992) have analysed the replication of the broad host range prolate-headed phage c6A and found that infection of L lactisC6 caused inhibition of culture growth within 10 min and cell Iysis after 25 min. DNA synthesis was detected after 6-8 min and stayed constant until cell Iysis. Using 3H- thymidine-Iabelled host DNA, it could be shown that degradation of the host DNA occurred after 4·6 min. The breakdown products were incorporated into the phage DNA. Molecular studies with the Lb casei phage PI-1 showed that it used unmodified host RNA polymerase to transcribe its en- tire genome. Early genes were expressed within the first 20 min, whereas the late genes were expressed after 40 min (Stet- teret al, 1978).
Following the synthesis of the phage macromolecular components, the phage
Reference
1
1
1
1
1
1
1
Organism Latent Burst
period (min) size
Lactococcus 10-140 10-400
Lactobacillus 40-75 80-300
Leuconostoc
mesenteroides 28 41
Leuconostoc oenos 60 16-20
Keogh,1973 Klaenhammer, 1984 Sechaud et al, 1988
Neve and Teuber, 1991 Arendt et al, 1990
194 EK Arendt et al
particles are assembled by a mechanism which has not yet been elucidated. At the end of the latent period the peptidoglycan layer of the host cell is Iysed by phage en- coded lysozyme Iike Iysin. Lysins of LAS phages have been isolated and character- ised in detail (see table III). .
CLASSIFICATION OF LAB PHAGES
Much effort has been focussed on the de- velopment of a coherent classification sys- tem for LAS phages. These taxonomie studies are very important for the following reasons: they provide an insight into the relationship between phages; they may allow identification of the origin of the phages disturbing fermentation processes;
they provide knowledge of the genetic characteristics of phages, which can th en be useful in predicting the potential utility of phage resistance mechanisms. Taxo- nomie characterisations of phage are based on morphology, DNA homology, protein composition and serological analy-
Table III.Genes/elements clonedlrom LAB phages.
sis. Since it is beyond the scope of this ar- ticle to review the characterisation and classification of LAS phages, the reader is directed to a number of comprehensive publications dealing with this topic (Lahbib- Mansais et al, 1988; Neve et al, 1989; Jar- vis et al, 1991; Soizet et al, 1992).
BACTERIOPHAGE GENOME CHARACTERISATION
Ali LAS phages examined to date possess double-stranded DNA. The majority of these appear to have cohesive ends al- though a few phages show a circularly per- muted structure with terminal redundancy.
This is based on the following criteria : the presence of submolar fragments atter re- striction analysis; evidence for the pres- ence of a pac site at which packaging of the phage genome is initiated; the absence of cohesive ends; and homology between the submolar fragment containing the terminal redundancy and the area of the genome close to the pac site. The G+C
Gene/Element Phage Reference
Lactococcus Lysin
Minor structural proteins Major capsid protein Undelined ORF LIai methylase
BK5- T promoter inhibitor Phage encoded resistance and phage origin 01replication
0vML3 F4-1 F4-1 07-9 050 BK5-T 050 Lactobacillus
Lysin
Phage structural proteins
LL-H LL-H Lysin
Insertion sequence ISL 1
mv1 0FSV
Shearman et al, 1989 Kim and Batl, 1991a,b Kim and Batl, 1991a,b Kim and Batl, 1991c Hilletai, 1991b Lakshmidevi et al, 1990 Hillet al, 1990
Trautwetler et al, 1986 Trautwetler et al, 1986 Alatassova et al, 1987 Boizet et al, 1990
Shimizu-Kadota et al, 1985
contents of the LAB phages are as fol- lows: Lactococcus (37-39 %); Lb plantar- um (37%) and Lb casei (45-48%). The genome lengths of LAB phages are su m- marised in table IV. Genetic determinants which have been cloned from phages of LAB are summarised in table III.
LYSOGENIC PHAGES OF LACTIC ACID BACTERIA
Lytic phages are of major concem in dairy fermentations, since they are primarily re- sponsible for the disruption of fermentation processes. However, there is also signifi- cant interest in phages which can enter an alternative relationship with their host, the Iysogenic cycle. In this case, the phage ge- nome integrates into the chromosome after injection into the target cell, usually by re- combination between 2 sites termed attP (phage attachment site) and attB (bacterial attachmentsite) on their respective ge- nomes. Little information is available re- garding the molecular or genetic basis for control and maintenance of the Iysogenic relationship in LAB hosts.
Table IV. Genome length of LAB phages.
Organism Genome
length (kb)
Lactococcal phage Smail isometric Large isometric Prolate-headed Lactobacillus phage
L aelbtûeckl!
29-40 53-55 18-22 34-41 75
Streptococcus thermophilus phage 33.8-44.2 Leuconostoc mesenteroides phage 27.4 Leuconostoc oenos phage 25.3
The study of Iysogeny in LAB is very worthwhile for a number of reasons. Lyso- genic hosts can serve as a reservoir of lyt- ic phage in dairy plants. One classical ex- ample demonstrating this was presented by Shimizu-Kadota et al (1983), who showed that a phage which was derived from a Iysogenic Lb casei host, was able to infect a Lb casei strain used in a yakult fermentation.
Lysogeny can also serve as a model system for the analysis of the regulation and control of phage gene expression. The information obtained from such a study cou Id be used, for example, in the con- struction of integration vectors. Chopin et al (1989) were able to generate a chromo- somal integration vector harboring a frag- ment of lactococcal prophage DNA, which was homologous to a resident prophage in the host chromosome. Integration was achieved by homologous recombination.
Temperate phages are also capable of me- diating transduction which can be exploited to transfer plasmid- and chromosomally- encoded traits and which may also prove useful in developing chromosomal maps for various members of the LAB (Fitzgerald and Gasson, 1988; Davidson et al, 1990).
Transduction of lactose-fermentation ability and proteolytic activity has already been reported (McKay and Baldwin, 1974; Gas- son, 1983; Fitzgerald and Gasson, 1988).
CHARACTERISATION OF TEMPERATE PHAGES AT THE MOLECULAR LEVEL
Lakshmidevi et al (1988) have character- ised the temperate L lactis subsp cremoris phage BK5- T in detail, The unit genome size of this phage is 37.6 kb, and it was shown to be circularly permuted with termi- nai redundancy and therefore DNA pack- aging is carried out by a headfull mecha- nism, giving rise to considerable variation
196 EK Arendtet al
in genome size (39.7-46 kb). Its Iysogenic host is L laetis subsp eremoris BK5 but the phage is also capable of propagating Iytically on L laetis subsp eremoris H2.
When phage BK5-T was grown on this Iyt- ic host, it lost its ability to Iysogenize its Iy- sogenic host, a feature which was Iinked to a deletion of 0.6-2.5 kb in a specifie re- gion of the genome, at a location separate from the attP site. Lakshmidevi et al (1990) also isolated 5 promoters from phage BK5-T and they couId also identify a region (621 bp) on the phage genome which was responsible for the inactivation of 3 of these.
The temperate lactococcal phage Tuc2009 isolated fromLaetoeoeeeus lectis subsp eremoris UC509 by induction with mitomycin Chas been analysed in sorne detail, The phage has a small isometric- head (52 nm), possesses a non-contractile tail (152 nm) and a base plate (16 nm across). Electronmicroscopic examination of Laetoeoeccus lactis subsp cremoris UC526 host cells mixed with phage Tuc2009 revealed that the phage particles attached evenly over the cell surface.
When the structural proteins of the phage were analysed by SDS-PAGE 2 major pro- teins with a molecular weight of 30 and 21.5 kDa were identified. The first 15 and 8 amino acids, respectively, were identi- fied by N-terminal sequencing (Arendt, un- published results). Restriction analysis showed that phage Tuc2009 has a ge- nome size of '" 40 kb. No evidence could be obtained for the presence of cohesive sites(cos). More-over, the presence of the submolar fragments in restriction enzyme digests of the phage DNA suggested that packaging occurs though a headfull mech- anism yielding circularly permuted ge- nomes. The site at which packaging starts (pac), and the attachment site(attP), have been localised to particular restriction frag- ments. Based on DNA-RNA and DNA- DNA hybridization studies, early and late
gene expression functions could be attrib- uted to specific regions of the phage ge- nome. Phage-specific DNA could be de- tected 40 min after infection and there was a consistent increase up to 80 min, and thereafter the concentration of detectable DNA decreased presumably due to pack- aging. While low levels of transcription products were detected 28 min after infec- tion of the host, there was not a significant increase in the level of RNA until 48 min.
Using various restriction fragments span- ning the entire Tuc2009 genome to probe the RNA sampies, it was possible to identi- fy early and late functions on the phage re- striction map (Fitzgerald, unpublished re- sults).
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